This invention relates to an assay for the detection of mammalian heparanase activity present in a variety of mammalian tissues, cells and bodily fluids, including serum and both normal and cancerous cells and tissues, and which may be used for the determination of mammalian heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of metastatic potential, and for the development of specific inhibitors of heparanase activity and metastasis.
The invention also relates to a method for the purification of mammalian heparanase, in particular human platelet heparanase, and to the purified mammalian heparanase obtainable by this method. The availability of purified mammalian heparanase such as human platelet heparanase will facilitate the development of inhibitors of mammalian heparanase activity, the production of monoclonal antibodies against the enzyme, and the identification of amino acid and cDNA sequence of the enzyme for the ultimate cloning and the expression of the enzyme.
Important processes in tissue invasion by blood-borne malignant tumour cells and leukocytes include their adhesion to the luminal surface of the vascular endothelium, their passage through the vascular endothelial cell layer and the subsequent degradation of the underlying basal lamina and extracellular matrix (ECM) with a battery of secreted and/or cell surface protease and glycosidase activities (Nakajima et al, 1983; Schmitt et al, 1992; Vlodavsky et al, 1992). The basal lamina and underlying connective tissue stroma consist predominantly of a complex network of fibronectin, laminin, collagen type IV and vitronectin, each of which interact with heparan sulphate (HS) side chains of heparan sulphate proteoglycans (HSPG) embedded within the matrix (Yurichenco and Schittny, 1990). HS chains generally consist of clusters of sulphated disaccharide units (predominately N-sulphated glucosamine linked 1xe2x86x924 to xcex1-L-iduronic acid residues) separated by lowly or non-sulphated regions (predominately disaccharide units of N-acetylated glucosamine linked 1xe2x86x924 to xcex2-D-glucuronic acid) (Turnbull and Gallagher, 1990,1991). Cleavage of the HS chains by endoglycosidase or heparanase activity produced by invading cells may therefore assist in the disassembly of the ECM and facilitate cell migration. Heparanase activity has been shown to be related to the metastatic potential of murine and human fibrosarcoma and melanoma cell lines (Nakajima et al, 1983, 1986a,b, 1988; Ricoveri and Cappelletti, 1986). Furthermore, heparanase activity has been described in a number of tissues and cell types including rat liver (Gallagher et al, 1988; Hook et al, 1975), human placenta (Klein and Von Figura, 1979; Lider et al, 1989), human platelets (Hoogewerf et al, 1995; Oldberg et al, 1980; Oosta et al, 1982), cultured human skin fibroblasts (Klein and Von Figura, 1976), human neutrophils (Matzner et al, 1985, 1992), activated but not resting rat T-lymphocytes (Naparstek et al, 1984), normal and neoplastic murine B-lymphocytes (Laskov et al, 1991), human monocytes (Sewell et al, 1989) and human umbilical vein endothelial cells (Bartlett et al, 1995; Godder et al, 1991). Because the cleavage of HS appears to be essential for the passage of metastatic tumour cells and leukocytes through basement membranes, studies of heparanase inhibitors provide the potential of developing novel and highly selective classes of anti-metastatic and anti-inflammatory drugs (Coombe et al, 1987; Irimura et al, 1986; Parish et al, 1990; Vlodavsky et al, 1992; Willenborg and Parish 1988).
The expression of heparanase activity by platelets, metastatic tumour cells and circulating cells of the immune system has been related to their involvement in their diapedes and extravasation. Studies have shown that while the initial entrapment of metastatic tumour cells by the capillary endothelium is platelet-independent, platelet aggregation which occurs shortly afterwards can lead to platelet activation and degranulation, resulting in gap formation and retraction of endothelial cells exposing the underlying basement membrane to adhesion by the tumour cells (Tanaka et al, 1986; Crissman et al, 1985; Yahalom et al, 1985). Human platelets have been shown to contain high levels of heparanase activity, capable of degrading endothelial cell surface, tumour-derived and ECM-derived HSPG (Bartlett et al, 1995a, 1995b; Castellot et al , 1982; Hoogewerf et al, 1995; Wasteson et al, 1976, 1977; Yahalom et al, 1984; ) as well as free HS and heparin chains (Graham et al, 1995a and b; Oldberg et al, 1980; Oosta et al, 1982; Wasteson et al, 1976, 1977). To date, three separate mammalian cell heparanase activities have been reported: mouse melanoma B16 heparanase which cleaves HS only, human platelet heparanase which cleaves both heparin and HS and a mouse mastocytoma endoglucuronidase which was reported to cleave newly synthesised heparin precursor but not heparin or HS (Hoogewerf et al 1995; Nakajima et al, 1988; Thunberg et al, 1982). More recent studies have indicated that murine melanoma and macrophage extracts are in fact able to degrade both HS and heparin, however heparin was degraded to a lesser extent than by human platelet extracts (Graham and Underwood, 1996). Although Hennes et al (1988) reported that tumour-derived heparanase was able to degrade matrix HS but was unable to degrade endothelial cell surface HS, human platelets have been shown to degrade endothelial cell surface HS (Wasteson et al, 1977) which was shown by Gamse et al (1978) to be more heparin-like in structure. Thus, it is likely that the platelet heparanase, which is capable of degrading both heparin and HS, may play a critical role in degrading cell-surface HS in focal adhesion plaques, and aiding the extravasation of blood-borne cells.
Following partial purification of human platelet heparanase by Oldberg et al (1980), who determined that the enzyme was an endoglucuronidase which acted upon N-sulphated, iduronic acid-containing heparin biosynthetic intermediates, the enzyme was purified 240,000-fold to apparent homogeneity in 6% yield by a 6-column procedure (Oosta et al, 1982). The enzyme was a 134 kDa single subunit protein which was active towards 125I-heparin and was confirmed to be an endoglucuronidase. In subsequent studies, the platelet heparanase was shown to cleave endothelial cell surface heparin-like material which inhibited smooth muscle cell proliferation (Castellot et al, 1982) and to cleave the antithrombin III (AT III)-binding octasaccharide between GIcA-GIcNS3S (Thunberg et al, 1982), presumably resulting in the loss of the anticoagulant activity of heparin following its degradation (Oldberg et al, 1980). Although, the purified enzyme was shown to act towards an octasaccharide substrate, platelet extracts were also shown to degrade ECM-derived HS chains to 10 kDa (Yahalom, et al 1984) and to 5 kDa fragments (Freeman and Bartlett, unpublished observations), indicating the existence of specific structural motifs determining the site of cleavage. The size of heparin-cleavage products following platelet heparanase action has not been determined (Graham and Underwood, 1996; Oldberg et al, 1980; Oosta et al, 1982). Recently, however, Hoogewerf et al (1995) reported the 4100-fold purification of a human platelet heparanase activity in 8% yield which was shown to be an endoglucosaminidase that cleaved both heparin and HS principally to disaccharides following radiolabelling of the digestion products. The activity resided in the 8-10 kDa subunit CXC chemokines connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2 (NAP-2) which are members of the platelet basic protein family. In contrast, Graham and Underwood (1996) has since shown that the heparanase activity in human platelet extracts had a Mr of 40-60 kDa, and cleaved both HS and heparin, although the size of the degradation products was not determined.
The resolution of the reported differences in the molecular size (8 to 134 kDa), the substrate specificity (whether the enzyme is an endo-glucuronidase or endo-glucosaminidase activity), and the size of the substrate cleavage products (disaccharides or 5 to 10 kDa) requires purification of the enzyme(s) to homogeneity in high yield. While some studies of the substrate specificity of the platelet enzyme have been reported (Oldberg, et al 1980; Oosta et al, 1882; Thunberg, et al 1982), surprisingly little has been reported on the inhibition of the enzyme by sulphated polysaccharides (similar to the studies by Nakajima et al (1986a and b)) on the inhibition of tumour cell heparanase activity) apart from the use of some modified heparins and heparin itself to inhibit platelet degradation of ECM-associated HSPG (Eldor, et al 1987). This is especially surprising considering the potentially important role of platelets during the initial stages of tumour cell extravasation.
In order to purify and characterise mammalian heparanase activities and to screen for and develop effective heparanase inhibitors, a simple and rapid assay for heparanase activity is required. However, previous heparanase assays have been cumbersome and time consuming in both preparation of radiolabelled substrate and separation of degradative products from the uncleaved substrate. Frequently heparanase assays have involved the biosynthetic radio-labelling of ECM-associated HSPG and the detection of HS chain degradation by gel filtration analysis of radiolabelled material released from the ECM (Bartlett et al, 1995; Vlodavsky et al, 1992 and references within). Such an approach suffers from the main disadvantage that degradation of HS chains in an ECM involves the synergistic action of proteases which are required to expose the HS chains for subsequent heparanase attack (Bar-Ner et al, 1985, 1986; Benezra et al, 1992; Vlodavsky et al, 1988). Furthermore, most heparanase assays have required extensive degradation of the radiolabelled HS (or ECM-derived HSPG) substrate to allow separation of the degraded product from the substrate by gel filtration (Bartlett et al, 1995; Klein and Von Figura, 1979; Nakajima et al, 1986a; Vlodavsky et al, 1992), although cleavage of HS chains at a single site may be all that is required to allow passage of leukocytes and tumour cells through the basement membrane. Solid phase heparanase assays also have been developed where chemically and biosynthetically radiolabelled heparin and HS chains were attached to a solid support with release of radiolabel from the solid support being a measure of enzyme activity (Nakajima et al, 1986a; Oosta et al, 1982; Sewell et al, 1989). Such assays, however, suffer from the disadvantage that the immobilized substrate may be less accessible to the mammalian heparanase enzyme, and the coupling of the radiolabelled substrate to the solid support, via the substrate""s reducing terminus, is a complex and inefficient procedure. Previous studies have also radiolabelled both heparin and HS by iodination at naturally occurring glucosamine residues (Oosta et al, 1982) or by N-acetylation of the partially de-N-sulphated substrate (Nakajima et al, 1986a). Such procedures, however, may result in the masking, or the creation of new heparanase cleavage sites.
In one aspect, the present invention provides a method for detecting mammalian heparanase activity in a sample such as mammalian tissue, cells or bodily fluids.
In accordance with this aspect, the present invention provides a method for the detection of mammalian heparanase activity in a sample, which comprises the steps of:
(i) contacting the sample to be tested with a heparanase substrate for a time and under conditions sufficient for heparanase in the sample to degrade the heparanase substrate;
(ii) separating degradation products from undegraded or partially degraded heparanase substrate by binding the undegraded or partially degraded heparanase substrate with a heparan sulphate-binding protein; and
(iii) detecting separated degradation products to indicate heparanase activity in the sample.
In this aspect, the present invention also provides a kit for use in the detection of mammalian heparanase activity in a sample, which comprises, in compartmentalized form:
(i) heparanase substrate;
(ii) heparan sulphate-binding protein; and
(iii) optionally, directions for performing the method of the invention as broadly described above.
In another aspect, the present invention provides a novel procedure for the rapid purification of mammalian heparanase activity, more particularly human platelet heparanase activity, as well as purified mammalian heparanase prepared by this procedure.
According to this aspect, the present invention provides a method for the purification of mammalian heparanase from a heparanase-containing material, which comprises the steps of:
(i) contacting the heparanase-containing material with an immobilised lectin affinity chromatography matrix in the presence of detergent to bind heparanase activity to said matrix;
(ii) eluting a first purified heparanase-containing fraction from said matrix;
(iii) optionally contacting said first purified heparanase-containing fraction with an immobilised metal ion affinity chromatography matrix;
(iv) contacting said first purified heparanase-containing fraction with a dye-resin matrix to bind heparanase activity to said dye-resin matrix; and
(v) eluting a second purified heparanase-containing fraction from said dye-resin matrix.
In this aspect, the present invention also provides substantially purified mammalian heparanase prepared by the method described broadly above, in particular substantially purified human platelet heparanase characterised in that it has a native molecular mass (Mr) of about 50 kDa when analysed by gel filtration chromatography and by SDS-PAGE, and in that it degrades both heparin and heparan sulphate. The substantially purified human platelet heparanase of this invention degrades bovine lung and porcine mucosal heparin from 12 kDa to 6 and 4 kDa fragments respectively, and porcine mucosal heparan sulphate in a stepwise fashion from 22 kDa to 5 kDa fragments, by gel filtration analysis.
The heparanase purification method of this invention as broadly described above may also be used to purify other heparanases, including rat liver, rat 13762 MAT adenocarcinoma cell and human HCT116 colonic carcinoma cell heparanase. Substantially purified MAT and HCT cell heparanase have similar Mr to the human platelet heparanase discussed above, while substantially purified rat liver heparanase has a native Mr of about 45 kDa and has 3 bands on SDS-PAGE between 43 and 47 kDa.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
In one aspect, the present invention relates in particular to the detection of mammalian heparanase activity in serum, including serum of human cancer patients, as well as in a variety of tissue and cell homogenates or extracts, including for example human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates. The method may be used to determine heparanase activity in both human and non-human (e.g. rat and murine) sera, cell and tissue homogenates or extracts.
Preferably, the heparanase substrate is a labelled substrate, more preferably a radiolabelled substrate. The substrate is preferably heparan sulphate (HS) and particularly preferred substrates are 3H-porcine mucosal HS and 3H-bovine kidney HS. Preferably, porcine mucosal HS is partially de-N-acetylated and re-N-acetylated with 3H-acetic anhydride to yield the desired radiolabelled substrate.
As broadly described above, the sample to be tested is contacted with the heparanase substrate for a time and under conditions sufficient for heparanase, if any, in the sample to degrade the substrate. Suitable incubation times and conditions can be readily determined without undue experimentation. By way of example, incubation of the sample with the substrate may be carried out at a temperature in the range of 35xc2x0 C. to 40xc2x0 C., preferably about 37xc2x0 C., for a period of from 2 to 48 hrs, preferably about 16 hrs, at a pH in the range of from 4.2 to 7.5, preferably about pH 5.1. Preferably, the incubation step is performed in the presence of bovine serum albumin, N-acetylmannosamine and/or a detergent such as Triton X-100. As described in detail below, heparanase activity under suitable incubation times and conditions degrades the preferred porcine mucosal heparan sulphate substrate in a stepwise fashion from 22 to 17, then to 8 and finally to 5 kDa fragments or degradation products.
Separation of degradation products from undegraded or partially degraded heparanase substrate is carried out by binding to a heparan sulphate-binding protein. As used herein, the term xe2x80x9cheparan sulphate-binding proteinxe2x80x9d includes, but is not limited to heparin-binding proteins. A particularly preferred heparan sulphate-binding protein for use in the present invention is histidine-rich glycoprotein. More preferably, the histidine-rich glycoprotein (HRG) is chicken HRG, however other HRG including human HRG may also be used. It is to be understood, however, that the present invention extends to the use of other heparan sulphate- and heparin-binding proteins other than HRG including, for example, protease inhibitors such as antithrombin 111 and heparin cofactor 11; plasma lipoproteins such as apolipoprotein B-100 and apolipoprotein E; growth factors such as acidic and basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor and platelet-derived growth factor; extracellular matrix proteins such as laminin, collagen and elastin; enzymes such as lipoprotein lyase, superoxide dimutase, cathepsin G, elastase and the bacterial heparin lyases 1, 11 and 111; and other suitable binding proteins such as platelet factor 4, von willebrand factor, and xcex2-thromboglobulin. The present invention also extends to peptides derived from these heparan sulphate- or heparin-binding proteins particularly HRG, which contain the heparan sulphate- or haparin-binding region.
Preferably, the separation is carried out by binding to an immobilised heparan sulphate-binding protein. Any suitable method of immobilisation may be used, however it is presently preferred to immobilise HRG or other suitable binding protein by binding to agarose, more particularly Sepharose, beads. in this preferred aspect, the HRG-Sepharose beads may be loaded in a column, and the incubation mixture applied to and washed through the column to bind undegraded or partially degraded heparanase substrate, with degradation products being unbound or bound less efficiently to the HRG-Sepharose beads. Alternatively, the HRG or other suitable binding protein may be added to an incubation mixture to bind to undegraded or partially degraded heparanase substrate complex, and the complex may then be separated, for example by immobilisation on a substrate (such as Immobilon or StrataClean) leaving degraded heparanase substrate fragments in the incubation mixture following removal of the immobilised complex, for example by centrifugation or filtration.
In the final step of the method of this aspect of the invention, separated degradation products are detected to indicate the presence of heparanase activity in the sample under test. This detection step may include quantitative and/or qualitative detection of the degradation products. Where a radiolabelled heparanase substrate such as a 3H-porcine mucosal HS is used, the detection step may consist of detection of radioactivity levels of unbound degradation products.
The present invention provides in this aspect a novel method for separating degradation products from the heparanase substrate by taking advantage of the decreased affinity of the cleaved heparanase degradation products for a heparan sulphate-binding protein such as HRG, particularly chicken histidine-rich glycoprotein (cHRG). Incubation mixtures are applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase degradation products. Heparanase activity has been determined for a variety of human, rat and murine cell and tissue homogenates. By way of example, highly metastatic rat mammary adenocarcinoma and murine melanoma cell lines had 4 to 10 times the heparanase activity compared to non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels as normal healthy adults.
This aspect of the present invention provides a new, simple and rapid quantitative assay for the detection of mammalian heparanase activity towards a natural radiolabelled substrate which can detect the minimal number of HS chain cleavages which are likely to occur in vivo, and which is based upon a novel principle, namely the loss of a protein binding site on HS chains following chain cleavage. In the assay, advantage is taken of an initial observation that the HS-binding plasma protein HRG masks heparanase cleavage sites on HS chains. Following heparanase digestion, radiolabelled HS fragments (products) are not bound by mini-columns of HRG-coupled Sepharose beads unlike the remaining intact and partially degraded substrate, allowing a rapid separation of the cleaved product from the substrate.
The assay of this aspect of the invention has several advantages over conventional assays in (a) ease of preparation of relatively large quantities of a radiolabelled natural substrate while maintaining the native structure, (b) allowing the rapid and simultaneous determination of a large number of samples and (c) allowing accurate quantification of heparanase activity by detecting single site cleavage of the substrate.
In another aspect, the present invention relates to the purification of mammalian heparanase from heparanase-containing material. The heparanase-containing material which is used as a starting material in the method of the present invention may be any source of mammalian heparanase activity, for example human colonic carcinoma HCT 116 cells, rat mammary adenocarcinoma 13762 MAT cells, or rat liver tissue. The present invention is, however, particularly directed to purification of human platelet heparanase, and a convenient source of this material is obtained by solubilisation of enzyme activity from a homogenate of frozen, washed human platelets.
The heparanase-containing material is subjected to immobilised lectin affinity chromatography, preferably concanavalin A-Sepharose chromatography, in the presence of detergent (such as Triton X-100) to bind the heparanase activity to the chromatography column. Elution of the bound heparanase activity provides a first purified heparanase-containing fraction.
Optionally, and preferably, the first purified heparanase-containing fraction is then subjected to immobilised metal ion affinity chromatography (IMAC), preferably using a matrix such as Zn2+-chelating Sepharose, which does not bind the hep-aranase activity but which removes other contaminating proteins. The first purified heparanase-containing fraction is then subjected to dye-resin chromatography, preferably in series with the IMAC matrix, to again bind the heparanase activity. Suitable dye-resin matrixes include a Blue-A agarose matrix, although other dyes such as Reactive Red may also be used as well as other matrix supports such as Sepharose and acrylamide. Elution of a second purified heparanase-containing fraction from the dye-resin matrix may be followed by octyl-agarose chromatography which again binds contaminating proteins without binding the heparanase activity.
Final purification of the second purified heparanase-containing fraction may be carried out by gel filtration chromatography, for example Superose 12 chromatography, optionally followed by concentration of the purified enzyme.
As previously described the product obtained in accordance with this aspect of the present invention is substantially purified mammalian heparanase, in particular substantially purified human platelet heparanase. The term xe2x80x9csubstantially purifiedxe2x80x9d as used in the present specification denotes that the product has been subjected to purification procedures such that the heparanase enzyme activity is at least 1000-fold, preferably at least 1,500-fold when compared with the activity of the heparanase-containing starting material.
Preferably also, the substantially purified mammalian heparanase is homogeneous, and preferably contains at least 75% by weight, even more preferably at least 85% by weight, and most preferably at least 95-99% by weight of the heparanase enzyme, relative to other proteins with which it is normally associated in its native form.
In accordance with this aspect of the present invention, human platelet heparanase has been purified to homogeneity from previously frozen, washed platelets. Human platelets have been shown to contain very high amounts of heparanase activity compared to metastatic tumour cells and other cell types when expressed as activity/mg of protein.
In one embodiment of this aspect of the method of this invention which is described in detail in the Example below, human platelet heparanase has been purified 1900-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The same purification procedure can be used to purify other mammalian heparanase activities, eg from human colonic carcinoma HCT 116 cells, rat mammary adenocarcinoma 13762 MAT cells and from rat liver. The human platelet heparanase, which degrades both heparin and HS, had a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS-PAGE. The enzyme degraded porcine mucosal HS in a stepwise fashion from 22 to 17, 10 and finally to 5 kDa fragments while bovine lung and porcine mucosal heparin were degraded from 12 kDa to 6 and 4 kDa fragments respectively by gel filtration analysis.